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Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at − 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL−1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 μL plasma and 2.5 μg brain tissue extracts).

A magnetic beads (MB)-involved amperometric immunosensor for the determination of ST2, a member of the IL1 receptor family, is reported in this work. The method utilizes a sandwich immunoassay and disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates built on the surface of carboxylic acid-microsized magnetic particles (HOOC-MBs) were used to selectively capture ST2. A biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP) was used to accomplish the sandwiching of the target protein. The immune platform exhibits great selectivity and a low limit of detection (39.6 pg mL ) for ST2, allowing the determination of soluble ST2 (sST2) in plasma samples from healthy individuals and patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in only 45 min once the immunoconjugates have been prepared. The good correlation of the obtained results with those provided by an ELISA kit performed using the same immunoreagents demonstrates the potential of the developed strategy for early diagnosis and/or prognosis of the fatal PDAC disease., This research was funded by Spanish Ministerio de Ciencia e Innovación. PID2019- 103899RB-I00, Spanish Ministerio de Ciencia, Innovación y Universidades, RTI2018-095672-B-I00, Comunidad de Madrid, S2018/NMT-4349, and AES-ISCIII/FEDER, PI20/00625

A sensitive and rapid method for the determination of the clinically relevant biomarker human endoglin (CD105) in serum samples is presented, involving a magneto-actuated immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Micro-sized magnetic particles were modified with a specific antibody to selectively capture the target protein which was further sandwiched with a secondary HRP-labeled antibody. The immunocomplexes attached to the magnetic carriers were amperometrically detected at SPCEs using the hydroquinone (HQ)/H2O2/HRP system. The magneto-actuated immunosensing platform was able to detect 5 pmoles of endoglin (in 25μL of sample, 0.2μM) in 30min providing statistically similar results to those obtained using a commercial ELISA kit for the determination of endogenous content of endoglin in human serum samples.

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5′-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

This work reports a new sensitive strategy for the determination of tau protein, a hallmark of Alzheimer's disease (AD), involving a sandwich immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) modified with a gold nanoparticles-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM) covalently immobilized onto electrografted p-aminobenzoic acid (p-ABA). The capture antibody (CAb) was immobilized by crosslinking with glutaraldehyde (GA) on the amino groups of the 3D-Au-PAMAM-p-ABA-SPCE, where tau protein was sandwiched with a secondary antibody labeled with horseradish peroxidase (HRP-DAb). Amperometry at −200 mV (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate was used to detect the immunocomplex formation. The great analytical performance of the immunosensor in terms of selectivity and low limit of detection (LOD) (1.7 pg mL−1) allowed the direct determination of the target protein in raw plasma samples and in brain tissue extracts from healthy individuals and post mortem diagnosed AD patients, using a simple and fast protocol.

A highly sensitive amperometric magnetoimmunoassay for rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum, is described. It uses a sandwich configuration involving selective capture antibody [antiLp(a)] and biotinylated detector antibody [biotin-antiLp(a)], and a streptavidin-HRP conjugate on carboxy-modified magnetic beads (HOOC-MBs). The resulting MBs bearing the sandwiched immunoconjugates were captured by a magnet placed under the working electrode surface of a disposable screen-printed carbon electrode and the extent of the affinity reaction was monitored amperometrically at −0.20 V (vs a silver pseudo-reference electrode) in the presence of hydroquinone as an electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The method exhibited a wide linear response range (from 0.01 to 0.5 μg mL−1), a detection limit of 4 ng mL−1, and an excellent selectivity over other serum components. The utility of the immunoassay was demonstrated by analyzing a reference serum containing a certified quantity of Lp(a). The performance of this magnetoimmunoassay compares favorably to that of an integrated amperometric immunoassay described earlier.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor involved in tumor growth and metastasis by regulating genes involved in response to hypoxia. This work reports the first magnetic beads (MBs)-based electrochemical immunoassay for the determination of HIF-1α. The design involves a sandwich immunoassay using MBs and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Magnetic immunoconjugates of carboxylic acid-microsized magnetic particles (HOOC-MBs) modified with a specific antibody were used to selectively capture the target protein which was sandwiched with a biotinylated secondary antibody further conjugated with a streptavidin peroxidase conjugate (Strep-HRP). The great analytical performance of the immunoassay in terms of selectivity and low limit of detection (76 pg mL−1) for HIF-1α allows the determination of the endogenous amount of this biomarker in 0.5 μg raw cancer cells lysates cultured in normoxia or induced-hypoxia with CoCl2. In addition, the developed immunosensing approach was employed to analyze HIF-1α in saliva samples with results that agree with those provided by an ELISA kit involving the same immunoreagents.

A highly sensitive magnetoimmunosensor for the determination of human C-reactive protein (CRP) is described in this work. A sandwich format involving covalent immobilization of the capture antibody (antiCRP) onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), the antigen–antibody reaction and incubation of the modified MBs with a biotynilated antibody (biotin-antiCRP), is used. Furthermore, an incubation step with a Streptavidin HRP (Strp-HRP) conjugate is employed to allow monitoring of the affinity reaction. The resulting modified MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE) and the amperometric responses are measured at −0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate. The CRP magnetoimmunosensor exhibited a wide range of linearity between 0.07 and 1000 ng mL−1 with a low detection limit of (0.021 ± 0.005) ng mL−1. The magnetoimmunosensor was successfully applied to the analysis of an international standard for CRP serum.

This work demonstrates the design and fabrication of an all cyclo-olefin polymer based microfluidic device capable of capturing magnetic beads and performing electrochemical detection in a series of gold electrodes. The size of chip is of a microscope slide and features six independent measuring cells for multianalyte detection purposes. The aim of this work is to show that rapid prototyping techniques can be instrumental in the development of novel bioassays, particularly in clinical diagnosis applications. We show the successful determination of troponin-T, a cardiac disease marker, in the clinically relevant range of 0.05-1.0 ng/mL. This methodology achieves a detection limit of 0.017 ng/mL in PBS solutions, and is capable of detecting less than 1 ng/mL in a 1:50 human serum dilution.

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at -0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H2O2 was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

Label-free electrochemical impedance immunosensors for the detection and quantification of Escherichia coli (E. coli) using self-assembled monolayers (SAMs)-modified gold screen-printed electrodes (AuSPEs) were developed. Two different immunosensor configurations were tested and compared. In the first one, the immunosensing design was based on the covalent immobilization of anti-E. coli at AuSPEs using the homobifunctional cross-linker 3,3'-dithiobis[sulfosuccinimidylpropionate] (DTSSP). The other one was based on the immobilization of the thiolated antibody onto the electrode surface. In both cases, the evaluation of the developed immunosensors performance was accomplished through the monitoring of the electron-transfer resistance detected by electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)(6)(3-)]/[Fe(CN)(6)(4-)] as redox probe. The configuration using the thiolated antibodies gave rise to a better analytical performance, exhibiting a linear relationship between the increment in the electron-transfer resistance (DeltaR(et)) and the logarithmic value of the E. coli concentration in the 5-1.0 x 10(8) cfu mL(-1) range. The limit of detection achieved, with no need for preconcentration or pre-enrichment steps was 3.3 cfu mL(-1). The developed immunosensors showed a high selectivity against Staphylococcus aureus (S. aureus) and Salmonella choleraesuis (S. choleraesuis). The usefulness of the thiolated antibodies-based design for the rapid analysis (1h) of 10 cfu mL(-1)E. coli inoculated river and tap water samples was demonstrated.

The preparation of novel Staphylococcus aureus ( S. aureus ) amperometric immunosensing designs based on the covalent immobilization of RbIgG at gold electrodes using the heterobifunctional cross-linker 3,3-dithiodipropionic acid di( N -succinimidyl ester) (DTSP), are reported. Two different competitive immunosensing configurations have been tested and compared. In the first one, protein A-bearing S. aureus cells and HRP-labelled antiRbIgG compete for immobilized RbIgG binding sites, while in the second case HRP-labelled protein A was used. In both cases, the evaluation of the developed immunosensors performance was accomplished through the monitoring at 0.00 V (vs. Ag/AgCl) of the catalytic current originated after addition of hydrogen peroxide, using tetrathiafulvalene as redox mediator entrapped at the modified electrode surface by cross-linking with glutaraldehyde. Optimization of variables concerning the composition of the immunosensors as well as the detection conditions was carried out in 0.1 M NaAc/0.1 M NaCl buffer of pH 5.6. The configuration that employed antiRbIgG-HRP resulted in better analytical characteristics, with a detection limit of 1.4 × 10 4 cells mL −1 for S. aureus cells submitted to wall lyses by ultrasonic treatment. This immunosensor design was also evaluated using gold screen-printed electrodes in order to reduce the analysis time and cost. In this case, a limit of detection of 3.7 × 10 2 cells mL −1 and a dynamic range from 1.3 × 10 3 to 7.6 × 10 4 cells mL −1 was obtained. A RSD value of 10.5% was found for the responses to 9.6 × 10 3 S. aureus cells mL −1 obtained with seven different Au/SPEs-immunosensors. These disposable immunosensors were applied to the quantification of S. aureus in milk spiked at two concentration levels, 1.2 × 10 3 and 4.8 × 10 3 cells mL −1 , with good recoveries.

7 páginas, 8 figuras, 1 tabla -- PAGS nros. 8239-8245, Disposable magnetic DNA sensors using an enzyme-amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response obtained at −0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD = 7.1%, n = 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology, The financial support of Santander/Complutense Research Project PR 27/05-13953 and of the Spanish Ministerio de Educación y Ciencia Research Project CTQ2006-02743BQU is gratefully acknowledged. Oscar A. Loaiza acknowledges a pre-Ph.D. fellowship of the Universidad Complutense de Madrid. Susana Campuzano acknowledges a “Juan de la Cierva” contract to the Spanish Ministerio de Educación y Ciencia

The usefulness of tetrathiafulvalene (TTF) derivative self-assembled monolayers (SAMs) modified electrodes as platforms for the construction of electrochemical biosensors is evaluated by using a 4-mercaptotetrathiafulvalene (HSTTF) modified gold electrode for the preparation of a fructose electrochemical biosensor. The electroactive SAM enhances the electron transfer between the immobilized FDH and the electrode surface, the electrochemical oxidation of HSTTF being used to monitor fructose concentration. The amount of HSTTF employed to form the electroactive SAM (2.2 μg), the FDH loading (20.4 U), and the applied potential to the biosensor (+0.40 V) were optimized. The stability of the FDH-HSTTF-AuE was evaluated under several aspects, a low reproducibility between different biosensors being observed. Calculation of the apparent Michaelis–Menten constant indicated that the affinity of the enzyme for the substrate is practically non-affected by the immobilization procedure. A detection limit of 2.7 × 10−7 mol L−1 fructose was achieved. The reproducibility of the responses obtained with different biosensors was largely improved by forming mixed monolayers consisting of HSTTF and mercaptohexanol (MCH). The limit of detection calculated for the FDH-MCH/HSTTF-AuE biosensor is 4.2 × 10−7 mol L−1 fructose. The performance of this biosensor for the determination of fructose in real samples is evaluated by the analysis of a Cola soft drink sample.

This paper describes specific electrochemical enterobacteriaceae lac Z gene DNA sensors based on immobilization of a thiolated 25 base single stranded probe onto disposable screen printed gold electrodes (gold SPEs). Two configurations have been evaluated. In the first one, the capture probe was attached to the electrode surface through its SH moiety, while mercaptohexanol (MCH) was used as spacer for the displacement of nonspecifically adsorbed oligonucleotide molecules. The hybridization event between the probe and target DNA sequences was detected at −0.20 V by square-wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. The second genosensor configuration involved modification of gold high temperature SPEs with a 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) self-assembled monolayer (SAM). Moreover, 2-aminoethanol was used as blocking agent, and further modification with avidin allowed binding of the biotinylated enterobacteriaceae lac Z gene DNA probe. An enzyme amplified detection scheme was applied, based on the coupling of streptavidin-peroxidase to the biotinylated complementary target, after the hybridization process, and immobilization of tetrathiafulvalene (TTF) as redox mediator atop the modified electrode. The amperometric response obtained at −0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. Experimental variables concerning sensors composition and electrochemical transduction were evaluated in both cases. A better precision and reproducibility in the fabrication process, as well as a higher sensitivity were achieved using the biotinylated probe-based sensor configuration. A limit of detection of 0.002 ng/μL was obtained without any preconcentration step.

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self-assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A-bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3-mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H2O2, and it was co-immobilized onto the MPA-modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×105S. aureus cells mL−1), as well as a better repeatability and reproducibility of the measurements.

The construction and performance under flow-injection conditions of an integrated amperometric biosensor for hydrogen peroxide is reported. The design of the bioelectrode is based on a mercaptopropionic acid (MPA) self-assembled monolayer (SAM) modified gold disk electrode on which horseradish peroxidase (HRP, 24.3 U) was immobilized by cross-linking with glutaraldehyde together with the mediator tetrathiafulvalene (TTF, 1 μmol), which was entrapped in the three-dimensional aggregate formed. The amperometric biosensor allows the obtention of reproducible flow injection amperometric responses at an applied potential of 0.00 V in 0.05 mol L −1 phosphate buffer, pH 7.0 (flow rate: 1.40 mL min −1 , injection volume: 150 μL), with a range of linearity for hydrogen peroxide within the 2.0 × 10 −7 –1.0 × 10 −4 mol L −1 concentration range (slope: (2.33 ± 0.02) × 10 −2 A mol −1 L, r = 0.999). A detection limit of 6.9 × 10 −8 mol L −1 was obtained together with a R.S.D. ( n = 50) of 2.7% for a hydrogen peroxide concentration level of 5.0 × 10 −5 mol L −1 . The immobilization method showed a good reproducibility with a R.S.D. of 5.3% for five different electrodes. Moreover, the useful lifetime of one single biosensor was estimated in 13 days. The SAM-based biosensor was applied for the determination of hydrogen peroxide in rainwater and in a hair dye. The results obtained were validated by comparison with those obtained with a spectrophotometric reference method. In addition, the recovery of hydrogen peroxide in sterilised milk was tested.

The flow-injection and HPLC determination of the diuretic drug furosemide using pulsed amperometric detection (PAD) at cylindrical carbon fibre microelectrodes (CFMEs) is reported. Experimental conditions such as pH (6.5) and buffer concentration (0.05 mol l(-1) HPO4(2-)/H2PO4(-)) were optimized using square-wave voltammetry (SWV). Repetitive flow-injection amperometric measurements at +1.25 V for furosemide showed a continuous decrease in the peak current, probably as a consequence of the microelectrode surface fouling. However, a suitable amperometric detection of furosemide was achieved using a PAD program consisting of a two-step potential waveform with alternating anodic and cathodic polarization. The anodic (detection) potential was +1.25 V (time of application 0.1 s), and the cathodic (cleaning) potential was -0.20 V (t=0.2 s). A linear calibration graph was obtained for furosemide in the 5.0 x 10(-7)-1.0 x 10(-4) mol l(-1) concentration range, with a limit of detection of 1.7 x 10(-7) mol l(-1). HPLC-PAD at carbon fibre microelectrodes was used for the determination of furosemide in the presence of several thiouracil drugs and oxytetracycline (OTC). The mobile phase selected was a 25:75 acetonitrile:5.0 x 10(-3) mol l(-1) NaH2PO4 (pH 5.0) mixture. A linear calibration graph was obtained for furosemide in the 1-100 microM range, with a limit of detection of 0.55 microM. The usefulness of this method for the determination of furosemide in real samples was evaluated by performing the analysis of commercial milk samples spiked with furosemide at a concentration level of 4.5 x 10(-7) mol l(-1) (150 ng ml(-1)), as well as with other thiouracil drugs and OTC. A mean recovery of 95+/-5% furosemide was obtained.

A voltammetric method for the determination of the antibiotic oxytetracycline (OTC) in food samples is reported. Carbon fiber microelectrodes (CFMEs), which allow voltammetric measurements to be performed in a small volume (1 mL) of the analyte extract from the samples, are employed. Repeatable electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. Under the optimized square-wave conditions, a linear calibration plot for OTC was obtained in the 1.0×10−6–1.0×10−4 mol L−1 range, with a detection limit of 2.9×10−7 mol L−1 (150 ng mL−1) OTC. The determination of OTC by a flow-injection method with amperometric detection using a homemade flow cell specially designed to work with CFMEs, was also evaluated using pure acetonitrile as the carrier. The SW voltammetric method was applied to the determination of OTC in spiked milk and eggs samples, at 100 ng mL−1 and 200 ng g−1 levels, respectively. The procedure involved the extraction of the analyte in ethyl acetate, evaporation of the solvent and reconstitution of the residue in acetonitrile −5.0×10−4 mol L−1 tetrabutylammonium perchlorate medium. Recoveries of 96±8 and 91±8% were obtained for milk and eggs, respectively, by applying the standard additions method.

Gold disk microelectrodes (AuMEs, 50 μm O) have been used for the determination of the fungicide disulfiram (DSF) at low concentration levels by differential pulse adsorptive stripping voltammetry (dp-AdSV). The AuMEs were fabricated in the laboratory and characterized by scanning electron microscopy and cyclic voltammetry. The AuME was pretreated daily by polishing with 3-μm diamond powder for 60 s, and by applying successively potentials of −0.4, −0.8, −1.0, −1.2 and −1.5 V for 30 s each in 0.1 mol L−1 H2PO4−/HPO42− (pH 6.0). In between measurements, the application of a potential of −1.5 V for 30 s was only necessary. The DSF adsorption on the AuME surface allows its determination at trace levels by dp-AdSV using low supporting electrolyte concentrations (3.0 mmol L−1) with an accumulation time of 120 s, a pulse amplitude of 50 mV, and a scan rate of 50 mV s−1. A detection limit of 6.3×10−8 mol L−1 and a RSD of 1.3% at a 5.0×10−7 mol L−1 DSF concentration level (n=10) were obtained with a two-electrode system and no stirring during the deposition step. The effect of the presence of several potential interferences on the DSF stripping signal has been tested. The developed method has been applied to the determination of DSF in spiked pea seeds with good results.

An amperometric flow-injection method for the determination of antithyroid drugs such as 6-methyl-2-thiouracil (MTU) using a carbon fiber cylindrical microelectrode-based detector is reported. A home-made flow-cell specially adapted for working with cylindrical microelectrodes ranging between 4 and 10 mm in length was used for this purpose. Methanol containing 0.05 mol l −1 tetrabutylammonium perchlorate as the supporting electrolyte was employed, and a potential of +1.6 V was selected for the amperometric detection of MTU. No cleaning or electrode surface regeneration of the fiber was necessary during the whole working day. A limit of detection of 2.6×10 −7 mol l −1 (37 μg MTU l −1 ) was achieved. The method was applied to the determination of MTU in spiked feed samples (peas and corn flour), at the 142 μg g −1 level. The procedure involved the extraction of the drug in methanol, a clean-up step using Florisil cartridges and the use of the standard additions method. Recoveries of 81±6 and 85±8% were obtained for peas and corn flour, respectively.

A novel strategy for the construction of a disposable integrated amperometric immunosensor for the sensitive and rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum is reported. The approach uses a sandwich format involving the covalent immobilization of selective capture antibodies (antiLp(a)) on the surface of N-[N α,N α-bis(carboxymethyl)-lysine]-12-mercaptododecanamide (HS-NTA)-modified screen-printed carbon electrodes (SPCEs). After a blocking step with skimmed milk, the modified antiLp(a)-SPCEs were incubated with a mixture solution containing the target analyte and a fixed concentration of a specific biotinylated antibody (biotin-antiLp(a)) and a streptavidin-horseradish peroxidase (HRP) (Strep-HRP) conjugate. The amperometric responses of the resulting immunosensor at −0.10 V (vs an Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, were used to monitor the extent of the immunoreactions. The developed methodology exhibited a wide range of linearity between 0.02 and 10 μg mL−1, a low detection limit (LOD) of 8 ng mL−1, and a great selectivity against other serum components. The usefulness of the Lp(a) immunosensor was demonstrated by analyzing spiked serum samples as well as a reference serum containing a certified Lp(a) content.

Cylindrical carbon fiber microelectrodes (CFMEs) (8 μm in diameter, 8 mm in length) are used to develop a voltammetric method for the determination of the thyreostatic drug methylthiouracil (MTU) in animal feed samples. Methanol, containing 0.05 mol L−1 tetrabutylammonium perchlorate as supporting electrolyte, was selected as working medium because of the high MTU solubility in this solvent, which is also used for the extraction of MTU from real samples. Reproducible electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. The net-current square-wave (SW) oxidation signal of MTU was selected to be exploited analytically. Under the optimized SW conditions, a linear calibration plot for MTU was obtained in the 1.0×10−6–1.0×10−4 mol L−1 range, with a detection limit of 3.8×10−7 mol L−1. The SW voltammetric behavior of other thyreostatic drugs was checked. A simple extraction procedure with methanol, followed by a clean up step, was used for the determination of MTU in a commercial dry fodder sample spiked with this drug at the 500 μg g−1 level. The detection limit in this type of sample was found to be 8.6×10−6 mol L−1.

The electroanalytical behavior of the reduction of the herbicides aziprotryne (2-azido-4-isopropylamino-6-methylthio-1,3,5-triazine) and desmetryne (4-isopropylamino-6-methylamino-2-methylthio-1,3,5-triazine) in oil-in-water emulsions is reported. This medium allows the differential pulse polarographic determination of these s-triazines directly from their sample extracts in an appropriate organic solvent. Sodium pentanesulfonate was chosen as the most suitable surfactant to be used as emulsifying agent, whereas ethyl acetate was selected as the organic solvent to form the emulsions. The peak current was maximum in a 0.3 mol L(-1) HClO4 medium of the continuous aqueous phase for aziprotryne, and at pH 3.0 for desmetryne, and the potential became more negative as the pH increased for both herbicides. The limiting current is diffusion controlled and the electrode process is irreversible. Four electrons are involved in the overall electrochemical reduction process as determined by controlled potential coulometry, whereas the alpha n(a) values suggested that two electrons are involved in the rate-determining step. Using differential pulse polarography, aziprotryne and desmetryne can be determined in the emulsified medium over the concentration ranges 1.0 x 10(-7)-1.0 x 10(-4) mol L(-1), with limits of detection of 4.5 x 10(-8) mol L(-1) and 6.6 x 10(-8) mol L(-1), respectively. The method was applied to the determination of aziprotryne and desmetryne in spiked irrigation water. At concentration levels of 6.0 x 10(-7) mol L(-1) aziprotryne and 4.0 x 10(-7) mol L(-1) desmetryne, recoveries of 94 +/- 3% and 94 +/- 4%, respectively, were obtained after preconcentration on Sep-Pack C18 cartridges. Finally, partial least-squares regression (PLSR) has been used for treatment of the polarographic data obtained from mixtures of aziprotryne, desmetryne and simazine in oil-in-water emulsions. The size of the calibration set was of 29 samples by ninety two current measurements at different potentials. Prediction of the herbicides concentration within the range 1.0 x 10(-6)-1.0 x 10(-5) mol L(-1) was possible.

A rapid magnetoimmunosensor for the simulta- neous determination of two cardiac biomarkers, amino- terminal pro-B-type natriuretic peptide (NT-proBNP) and C-reactive protein (CRP), in human serum is de- scribed. Specific capture antibodies were covalently im- mobilized onto carboxylic acid-modified magnetic beads. The quantification of NT-proBNP and CRP was per- formed by using indirect competitive and sandwich con- figurations, respectively, and horseradish peroxidase-la- beled tracers. The use of dual screen-printed carbon elec- trodes allowed the achievement of simultaneous inde- pendent amperometric readout for each cardiac biomark- er. The developed methodology showed very low limits of detection (0.47 ng mL � 1 ). An international standard for CRP serum spiked with NT-proBNP was analyzed to evaluate the usefulness of the magnetoimmunosensor.

An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5′ end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg μL −1 and 10 pg μL −1 were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.

A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL−1) and a detection limit of 0.02 ng mL−1, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum samples.

The possibility of applying adsorptive stripping voltammetry (AdSV) in dispersed media has been evaluated. Sensitive methods for the determination of the herbicide terbutryn at nanomolar levels by AdSV at the hanging mercury drop electrode in micellar and emulsified media are proposed. The anionic surfactant sodium pentanesulfonate was chosen as the most suitable agent for micellar solutions and oil–water emulsions. On the other hand, ethyl acetate was used as the organic solvent to form terbutryn emulsions. A systematic study of the various experimental parameters that affect the stripping response was carried out by differential pulse voltammetry. When working in a 0.1 mol L−l perchloric acid medium with an accumulation potential of −0.60 V, terbutryn could be determined over the 6.0 × 10−9−4.0 × 10−7 and 6.0 × 10−9−2.0 × 10−7 mol L−1 ranges in micellar (50s accumulation time) and emulsified (70s accumulation time) media, respectively. The limits of detection were 2.2 × 10−9 and 1.1 × 10−9 × mol L−1, and the relative standard deviations (n = 10) were 6.3 and 4.3% in micellar and emulsified media, respectively. The effect of other coexisting herbicides on the terbutryn stripping peak was evaluated. Finally, the developed method in emulsified medium was applied to the determination of terbutryn in spiked tap waters.

A polarographic study of the reduction of the herbicide simazine in micellar solutions and oil-water emulsions is reported. The anionic surfactant sodium pentanesulphonate was chosen as the most suitable agent for micellar solutions and emulsions. In micellar solutions two reduction peaks were observed in differential-pulse polarography (DPP) below pH 2.0, whereas only one peak was obtained above pH 2.0. Ethyl acetate was chosen as the organic solvent to form simazine emulsions. Unlike in micellar solutions, DP polarograms of simazine in oil-water emulsions showed only one peak even at pH values lower than 2.0, suggesting that pesticide hydrolysis is hindered in the emulsified medium. The limiting current is diffusion-controlled and the electrode process is irreversible. The diffusion coefficient of simazine in emulsified medium was 8.85 × 10 −6 cm 2 s −1 , and the α n a and K ° f values were 0.82 and 8.03 × 10 −14 cm s −1 respectively. Using DPP, simazine can be determined over the concentration ranges 8.0 × 10 −7 −4.0 × 10 −5 mol l −1 . The limit of detection was 2.2 × 10 −7 mol l −1 . The effect of the presence of other herbicides belonging to the s -triazine family on the simazine reduction peak was studied. The method was applied to the determination of simazine in spiked irrigation water with good recoveries.

Sensitive methods for the determination of the herbicides methoprotryne and terbutryn at nanomolar levels, by adsorptive stripping voltammetry at a hanging mercury drop electrode, are described. The electrocapillary curves, and cyclic voltammograms, demonstrate the adsorption of these compounds on the mercury electrode. A systematic study of the various experimental parameters that affect the stripping response was carried out by differential pulse voltammetry. The working medium chosen was 0.1 mol l–1 perchloric acid. By using an accumulation potential of –0.70 V and a 180 s accumulation time, the limits of detection were 5.2 × 10–10 and 2.4 × 10–9 mol l–1 for terbutryn and methoprotryne, respectively, and the relative standard deviations (n= 10) were 3.1% and 5.0% at concentration levels of 5.0 × 10–9 mol l–1 terbutryn and 1.0 × 10–8 mol l–1 methoprotryne, respectively. The degree of interference from some other herbicides on the differential pulse stripping signal for methoprotryne and terbutryn was evaluated. Finally, the methods were applied to the determination of terbutryn and methoprotryne in spiked irrigation and tap waters.

6 páginas, 4 figuras -- PAGS nros. 1225-1230, Disposable amperometric magnetoimmunosensors, based on the use of functionalized magnetic beads and gold screen-printed electrodes, have been developed for the selective detection and quantification of Streptococcus pneumoniae. A specific antibody prepared against a serotype 37 S. pneumoniae strain, selected by flow cytometry among seven anticapsular or antisomatic antibodies, was linked to Protein A-modified magnetic beads and incubated with bacteria. The same antibody, conjugated with horseradish peroxidase, was attached to the bacteria and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene-modified gold screen-printed electrodes. The amperometric response obtained at −0.15 V vs. the silver pseudoreference electrode of the Au/SPE after the addition of H2O2 was used as transduction signal. Different assay formats were examined and the experimental variables optimized. The limits of detection achieved, without pre-concentration or pre-enrichment steps, were 1.5 × 104 cfu mL−1 (colony forming unit) and 6.3 × 105 cfu mL−1 for S. pneumoniae strains Dawn (serotype 37) and R6 (non-encapsulated), respectively. The developed methodology shows a good selectivity against closely related streptococci and its usefulness for the analysis of inoculated urine samples has been demonstrated. The total analysis time of 3.5 h from sampling to measurement, the possibility to prepare up to 30 sensors per day and the use of small amounts of test solution for S. pneumoniae identification, constitute important advantages that make the developed methodology a promising alternative for clinical diagnosis, This research was supported by grants from the Dirección General de Investigación Científica y Técnica (SAF2006-00390 and SAF2009-10824). The financial support of Santander/Complutense Research Project PR 27/05-13953, the Spanish Ministerio de Ciencia e Innovación Research Project CTQ2009-09351BQU, and the AVANSENS Programme from the Comunidad de Madrid (S2009PPQ-1642) are also gratefully acknowledged. S. Campuzano acknowledges a “Juan de la Cierva” research contract to the Spanish Ministerio de Ciencia e Innovación

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.

Screen printing of palladium-doped carbon strips results in the construction of disposable amperometric sensors for formaldehyde. These sensors rely on the strong electrocatalytic action of the dispersed palladium particles contributing to the reduction of formaldehyde. The electrocatalytic behaviour, sensor optimization and analytical performance are reported. The low detection limit (2 × 10–6 mol l–1) is accompanied by a wide linear range and high selectivity. Application for assays of various samples is demonstrated. The new single-use sensor strips should facilitate on-site environmental and industrial monitoring of formaldehyde.

Two flow injection (Fl) methods, conventional and stopped-flow, are described for the determination of formaldehyde based on its reaction with pararosaniline in the presence of sulphite ion followed by spectrophotometric detection at 570 nm. The calibration graphs are linear over the ranges 2.0–50 and 0.2–10 µg ml–1 of formaldehyde for conventional and stopped-flow Fl, respectively. The coefficient of variation was 6.3% at the 0.6 µg ml–1 level in the stopped-flow mode, the detection limit being 0.1 µg ml–1 of formaldehyde. Sampling rates of 41 and 18 samples h–1 were achieved in the conventional and stopped-flow modes, respectively, this being a major advantage of these methods. The effect of several organic and inorganic species was studied and only low relative molecular mass aldehydes were found to interfere. The stopped-flow method was applied to the determination of formaldehyde in air in work environments and its accuracy was confirmed by comparing the results with those obtained using the standard chromotropic acid method.